1. Depletion of NADP(H) due to CD38 activation triggers
endothelial dysfunction in the postischemic heart
Levy A. Reyesa,1
, James Bosletta,1
, Saradhadevi Varadharaja
, Francesco De Pascalia
, Craig Hemanna
,
Lawrence J. Druhana
, Giuseppe Ambrosioa,b
, Mohamed El-Mahdya
, and Jay L. Zweiera,2
a
Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State
University Medical Center, Columbus, OH 43210; and b
Division of Cardiology, University of Perugia School of Medicine, 06156 Perugia, Italy
Edited by Gregg L. Semenza, Johns Hopkins University School of Medicine, Baltimore, MD, and approved July 27, 2015 (received for review March 20, 2015)
In the postischemic heart, coronary vasodilation is impaired due to loss
of endothelial nitric oxide synthase (eNOS) function. Although the
eNOS cofactor tetrahydrobiopterin (BH4) is depleted, its repletion
only partially restores eNOS-mediated coronary vasodilation, indicat-
ing that other critical factors trigger endothelial dysfunction. There-
fore, studies were performed to characterize the unidentified factor(s)
that trigger endothelial dysfunction in the postischemic heart. We
observed that depletion of the eNOS substrate NADPH occurs in the
postischemic heart with near total depletion from the endothelium,
triggering impaired eNOS function and limiting BH4 rescue through
NADPH-dependent salvage pathways. In isolated rat hearts subjected
to 30 min of ischemia and reperfusion (I/R), depletion of the NADP(H)
pool occurred and was most marked in the endothelium, with >85%
depletion. Repletion of NADPH after I/R increased NOS-dependent
coronary flow well above that with BH4 alone. With combined NADPH
and BH4 repletion, full restoration of NOS-dependent coronary flow
occurred. Profound endothelial NADPH depletion was identified to be
due to marked activation of the NAD(P)ase-activity of CD38 and could
be prevented by inhibition or specific knockdown of this protein.
Depletion of the NADPH precursor, NADP+
, coincided with formation
of 2’-phospho-ADP ribose, a CD38-derived signaling molecule. Inhibi-
tion of CD38 prevented NADP(H) depletion and preserved endothe-
lium-dependent relaxation and NO generation with increased recovery
of contractile function and decreased infarction in the postischemic
heart. Thus, CD38 activation is an important cause of postischemic
endothelial dysfunction and presents a novel therapeutic target for
prevention of this dysfunction in unstable coronary syndromes.
ischemia reperfusion injury | endothelial nitric oxide synthase |
nitric oxide | endothelial dysfunction | tetrahydrobiopterin
Endothelial dysfunction is associated with a wide range of car-
diovascular diseases including hypercholesterolemia, diabetes,
atherosclerosis, hypertension, heart failure, and ischemic heart dis-
ease (1). In vivo coronary occlusion induces endothelial dysfunction
with decreased endothelial nitric oxide synthase (eNOS)-dependent
vasoreactivity, which persists upon reperfusion (2, 3). Persistent
diminished flow through the coronary arteries upon reperfusion can
lead to cardiac myocyte injury and heart failure (4). Endothelial
dysfunction is induced by the marked oxidant stress that occurs
following the onset of ischemia and reperfusion (I/R) (5).
Normally, vascular tone and coronary vasodilation are mod-
ulated by nitric oxide (NO). Synthesis of NO occurs within the
endothelium via eNOS, which uses L-arginine and O2 to form
NO and L-citrulline. This enzymatic process uses NADPH as the
source of reducing equivalents and requires Ca2+
/calmodulin,
flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN),
heme, and tetrahydrobiopterin (BH4) as cofactors. eNOS regulates
vasomotor tone and blood pressure by producing NO, which acti-
vates soluble guanylate cyclase in vascular smooth muscle, resulting
in vasorelaxation (6, 7).
In postischemic myocardium, the critical redox-sensitive eNOS
cofactor, BH4, is depleted, leading to eNOS uncoupling with
production of superoxide (O2
•–
) instead of NO (8, 9). The essen-
tial role of BH4 in NO production has made it a primary target
of therapeutic studies aimed at restoring endothelial function
following I/R (10). Preventing loss of and/or replenishing BH4
has proven beneficial, although recovery of eNOS function post-
BH4 replacement therapy remains incomplete (10, 11). Fur-
thermore, it remains unclear why BH4 salvage pathways fail to
adequately regenerate this critical cofactor. Thus, there is a great
need to identify other factor(s) that lead to eNOS dysfunction in
the postischemic heart and develop new therapeutic approaches
to restore endothelium-dependent vasodilatory function.
Studies were performed to characterize the previously un-
identified factors that trigger and contribute to postischemic en-
dothelial dysfunction. We observed that marked NADP(H) de-
pletion occurs, resulting in impaired eNOS function and limited
BH4 recycling through NADPH-dependent salvage pathways.
Tandem NADPH and BH4 supplementation resulted in complete
restoration of NOS-dependent coronary flow (CF). The profound
endothelial NADP(H) depletion was shown to be due to activation
of CD38. siRNA-mediated CD38 knockdown in endothelial cells
prevented NADP(H) depletion. CD38 inhibition preserved endo-
thelium-dependent CF, eNOS coupling, and NO generation, with
enhanced recovery of cardiac contractile function and decreased
infarction in the postischemic heart.
Results
Effects of I/R on Pyridine Nucleotides. Depletion of NADPH could
explain both the loss of eNOS function and failure to salvage
BH4 in the postischemic heart. It is not known if alterations
occur in the level of this critical reducing substrate required for
eNOS activity and BH4 resynthesis through dihydrofolate re-
ductase (10). The pyridine nucleotides NADP+
and NADPH as
well as NAD+
and NADH were measured in control hearts and
Significance
Vasodilation is impaired in the postischemic heart due to loss of
endothelial nitric oxide synthase (eNOS) function; however, the
central trigger of this endothelial dysfunction is unknown. We
observe that near total depletion of the eNOS substrate NADPH
occurs in postischemic endothelium, triggering eNOS dysfunction
and limiting rescue of its essential cofactor BH4. This NADPH
depletion was shown to be due to marked activation of the
NAD(P)ase activity of CD38 and could be prevented by its in-
hibition or genetic knockdown. Thus, CD38 activation with
pronounced NADPH depletion is first identified as a critical
trigger of postischemic endothelial dysfunction, and this pre-
sents a novel therapeutic target for the treatment of unstable
coronary syndromes and prevention of coronary disease.
Author contributions: L.A.R., J.B., L.J.D., G.A., M.E.-M., and J.L.Z. designed research; L.A.R.,
J.B., S.V., F.D.P., C.H., and M.E.-M. performed research; L.A.R., J.B., F.D.P., C.H., L.J.D.,
G.A., M.E.-M., and J.L.Z. analyzed data; and L.A.R., J.B., F.D.P., C.H., and J.L.Z. wrote
the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
L.A.R. and J.B. contributed equally to this work.
2
To whom correspondence should be addressed. Email: Jay.Zweier@osumc.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1505556112/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1505556112 PNAS Early Edition | 1 of 6
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2. hearts subjected to I/R. In control hearts, NADP+
and NADPH
levels were 23.5 ± 4.1 and 55.3 ± 4.3 nmol/g tissue, respectively,
whereas NAD+
and NADH levels were 564.3 ± 31.6 and 5.7 ±
0.2 nmol/g tissue, respectively (Fig. 1 and SI Appendix, Fig. S1). By
10 min of ischemia, NADP+
declined with concomitant increase in
NADPH, due to the reduced state of the myocardium that accom-
panies ischemia (12). By 30 min of ischemia, NADP+
and NADPH
both declined below preischemic levels, with a further dramatic
decrease following reperfusion. Importantly, although the total
NADP(H) pool does not change during early ischemia, depletion
occurs during later ischemia and following reperfusion, with NADP+
declining by ∼70% and NADPH levels declining by ∼50% by 30 min
of reperfusion (Fig. 1A). In contrast, a much lower decrease was
seen in the NAD(H) pool (∼25%; SI Appendix, Figs. S1 and S2).
Repletion of eNOS Cofactors/Substrates. To determine if post-
ischemic NADPH depletion contributed to the impaired NOS-
dependent CF, experiments were performed to test if NADPH
repletion could reverse this dysfunction. Liposomal NADPH ad-
ministration performed in hearts subjected to 30 min of ischemia
and 30 min of reperfusion (I/R) induced a prominent 30.9 ± 3.2%
rise in CF compared with baseline. When combined with BH4, CF
increased by 39.0 ± 5.7%, or >3 times the amount with BH4 alone
(10.0 ± 1.0%; Fig. 2A). Coadministration of the eNOS inhibitor,
nitro-L-arginine methyl ester (L-NAME), along with NADPH,
completely blocked the increase seen with NADPH administra-
tion with a 18.6 ± 6.2% decrease in CF (Fig. 2A). BH4 treatment
alone resulted in only ∼50% recovery of NOS-dependent CF.
NADPH treatment resulted in ∼85% recovery, whereas combined
BH4 and NADPH treatment resulted in full recovery (Fig. 2B).
To confirm that the observed NADPH depletion limits eNOS
activity, L-arginine to L-citrulline conversion assays were per-
formed in heart tissue with and without addition of NADPH. In
both control and postischemic tissues, NADPH addition was
required to observe maximum activity. With NADPH addition,
postischemic NOS activity was increased ∼fivefold to levels
similar to those in control tissue (SI Appendix, Fig. S3).
To verify that NADPH repletion-mediated restoration of post-
ischemic CF is eNOS-dependent, similar repletion studies were
performed in wild-type (WT) and eNOS−/−
mouse hearts. Although
liposomal NADPH repletion increased CF in WT hearts by ∼30%,
similar to that in the rat model, this effect was totally lost in
eNOS−/−
hearts. Thus, the NADPH-mediated increase in post-
ischemic CF is eNOS-dependent (SI Appendix, Fig. S4).
Endothelial Levels of NADP(H). Due to the marked impairment in
NOS-dependent CF, we hypothesized that the depletion of
NADP(H) in the endothelium would be more severe relative
to the depletion in the myocardium. To measure I/R effects on
pyridine nucleotides within the endothelium, we permeabilized
the endothelium of the heart as previously reported (13). NADP(H)
efflux from the endothelium in control hearts was measured with
0.45 ± 0.10 nmol detected, whereas with hearts subjected to
I/R only 0.069 ± 0.050 nmol of NADP(H) was detected (Fig. 2C).
Thus, I/R depletes the endothelial NADP(H) pool by almost 90%,
accounting for impairment in endothelial vasodilatory function,
which is restored by NADPH administration. Endothelial levels of
NAD(H) also trended downward after I/R, but the depletion was
not as severe as with the NADP(H) pool (SI Appendix, Fig. S5).
Expression, Activity, and Localization of CD38. NADP+
was prefer-
entially depleted during reperfusion, with the NADP(H) pool
almost totally depleted from the endothelium. Therefore, this
depletion was not simply due to oxidation of NADPH to NADP+
but to depletion of NADP+
as well. This implies that there must
be a process consuming NADP+
causing depletion of the NADP(H)
pool. CD38 preferentially uses NADP+
over NAD+
with >sixfold
Fig. 1. NADP(H) whole-heart measurements. (A) NADP+
and NADPH pre-
ischemia (PI), then during I/R. (B) Representative chromatograms showing
the NADP+
and NADPH peaks (ex. 330 nm; em. 460 nm) in nonischemic
control hearts and hearts with 30 min of ischemia (I) and 30 min of ischemia/
30 min reperfusion (I/R). For the entire chromatogram, see SI Appendix, Fig.
S2. *P < 0.05 compared with preischemic values (mean ± SEM; n = 5–7).
Fig. 2. Effect of eNOS cofactor/substrate repletion on CF and endothelial de-
pletion of NADP(H) after I/R. (A, Top) Tracings showing change in CF after ad-
ministration of control liposome (vehicle) or liposomal BH4 (50 μM), NADPH
(175 μM), NADPH (175 μM) + BH4 (50 μM), and NADPH (175 μM) + L-NAME
(1 mM), with the start of administration at time depicted by an arrow. (A, Bottom)
Graph of the mean changes in CF. NADPH elicited a dramatic rise in CF, much
greater than that of BH4. This increase in CF was NOS-dependent, as coinfusion
of L-NAME abolished NADPH-mediated vasodilation with vasoconstriction seen.
The effects of combined BH4 and NADPH infusion were additive. *P < 0.05
compared with vehicle; +
P < 0.05 compared with BH4 (mean ± SEM; n = 5).
(B) BH4 or NADPH treatment resulted in improved but incomplete restoration of
NOS-dependent CF, whereas combined, BH4 + NADPH resulted in complete
restoration. *P < 0.05 compared with vehicle; +
P < 0.05 compared with BH4
(mean ± SEM; n = 5). (C) Endothelial NADP(H) content was measured from the
effluent after endothelial permeabilization from hearts subjected to non-
ischemic perfusion (Control) or I/R. Endothelial NADP(H) levels were ∼90% lower
in hearts subjected to I/R compared with hearts subjected to nonischemic per-
fusion. **P < 0.01 versus untreated I/R group (mean ± SEM; n = 5–7).
Fig. 3. Multiple reactions of CD38. CD38 catalyzes several reactions in-
cluding (1) formation of the Ca2+
-liberating molecule 2’-P-cADPR; (2) hy-
drolysis of 2’-P-cADPR, forming 2’-P-ADPR; and (3) NAD(P)ase activity directly
generating 2’-P-ADPR from NAD(P)+
.
2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1505556112 Reyes et al.

3. lower Km and higher Vmax (14). We hypothesized that CD38, with
NAD(P)ase activity, which metabolizes NADP+
to 2’-phospho-
ADP ribose (2’-P-ADPR) and nicotinamide (NA), may be of im-
portance in this process of NADP(H) depletion (Fig. 3).
To determine the presence and location of CD38 in the heart,
immunoblotting and immunohistology were performed in control
hearts and hearts subjected to 30 min of ischemia or 30 min of
ischemia followed by 30 min of reperfusion (I/R). Control hearts
showed clear bands corresponding to CD38, and no change in
CD38 expression was seen with ischemia or I/R (Fig. 4A). Immu-
nohistochemistry demonstrated that high levels of CD38 are pre-
sent in the endothelium of the coronary vasculature and colocalize
with the endothelial markers von Willebrand factor (vWf) and
eNOS (Fig. 4B). Lower levels of staining were seen in cardiac
myocytes with an ∼eightfold lower intensity.
When CD38 activity was measured (as production of 2’-P-ADPR)
from these hearts, activity was low in control (nonischemic) hearts.
CD38 activity increased 2.2-fold after 30 min of ischemia and 5.5-
fold after 30 min of reperfusion (Fig. 4C and SI Appendix, Fig. S6A).
Furthermore, levels of 2’-P-ADPR in control hearts were barely
detectable (1.68 ± 0.42 nmol/g tissue) but greatly increased after I/R
to 9.11 ± 0.43 nmol/g tissue (SI Appendix, Fig. S6B).
CD38 Inhibition Salvages NADPH and Restores NOS-Dependent CF. As
CD38 activity and 2’-P-ADPR levels rise in the postischemic heart,
we would expect NADP+
to be consumed. To confirm the role of
CD38 in the process of NADP(H) depletion, experiments were
performed with the CD38 inhibitor, α-NAD (15). In hearts subjected
to I/R, administration of α-NAD immediately before the onset of
ischemia resulted in near total salvage of endothelial NADP(H) (Fig.
5A). In parallel with this, near total recovery of NOS-dependent CF
was seen (Fig. 5B). This confirms the role of CD38 in NADP(H)
depletion as well as the importance of NADP(H) levels in modu-
lating NOS-dependent CF in the postischemic heart (Fig. 5B).
CD38 Inhibition Improves BH4 Recovery. Due to the NADPH de-
pendence of the BH4 salvage pathway (9), we hypothesized that
CD38 inhibition with resultant NADP(H) preservation would
lead to enhanced BH4 salvage. In hearts pretreated with α-NAD,
∼twofold higher BH4 levels were observed after I/R compared
with those in untreated hearts (Fig. 5C). Thus, CD38 inhibition
enhanced both postischemic NADP(H) and BH4 salvage.
CD38 Inhibition Preserves NO and Decreases O2
•–
Production. EPR
spin trapping was performed to directly measure eNOS coupling
and function in the heart before and after I/R. NO production was
measured using Fe2+
-N-methyl-D-glucamine dithiocarbamate (Fe-
MGD) and O2
•–
using 5,5′-dimethyl-1-pyrroline-N-oxide (DMPO).
Following I/R, a >sixfold decrease in NO generation was seen, and
this was fully restored with α-NAD treatment (Fig. 5D and SI Ap-
pendix, Fig. S7). Following I/R, a large >10-fold increase in O2
•–
generation was seen compared with preischemic control, and this
was decreased ∼fourfold by the NOS inhibitor L-NAME. α-NAD
pretreatment blocked this NOS-dependent O2
•–
by ∼85% (SI Ap-
pendix, Fig. S8). Together these measurements demonstrate that
CD38 inhibition restored eNOS function and coupling.
CD38 Knockdown Enhances NADP(H) Recovery in Rat Aortic Endothelial
Cells. To further demonstrate the role of CD38 in NADP(H)
depletion, cultured rat aortic endothelial cells (RAECs) with or
without CD38 knockdown by shRNA were subjected to nor-
moxic treatment or hypoxia/reoxygenation (H/R). The RAECs
with CD38 knockdown showed preserved NADP(H) levels fol-
lowing H/R compared with RAECs receiving the control plasmid
lacking CD38 shRNA (empty plasmid) (SI Appendix, Fig. S9),
providing additional evidence that H/R-associated CD38 acti-
vation causes endothelial NADP(H) depletion.
Role of Oxidative Stress in CD38 Activation with Conversion of NADP(H)
to 2’-P-ADPR. I/R is associated with marked oxidative stress with
production of O2
•–
. Because CD38 activation has been reported to
be associated with oxidative stress (16–18), we questioned if I/R-
associated oxidant stress triggers CD38 activation. We observed
that CD38 is activated following I/R (Fig. 4C), and this leads
to a marked increase in 2’-P-ADPR levels in the myocardium.
We observed a >fivefold increase in 2’-P-ADPR from 1.68 ± 0.42 to
9.12 ± 0.44 nmol/g tissue following I/R (Fig. 6). When hearts
were treated with the superoxide dismutase (SOD) mimetic
Mn(III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), this
increase was largely blocked. The I/R-associated increase in
myocardial 2’-P-ADPR level was also totally abolished by the CD38
inhibitor α-NAD (Fig. 6). Thus, O2
•–
-derived oxidant stress is a
key trigger of CD38 activation with NADP(H) degradation.
Fig. 4. CD38 in the isolated rat heart. (A) Immunoblotting demonstrated that
CD38 expression was present in control heart tissue (control), and this was
unchanged by 30 min of ischemia (I) or 30 min of ischemia and 30 min of
reperfusion (I/R). (B) CD38 was found predominately within the vasculature of
isolated hearts. Confocal microscopy (60×) revealed CD38 colocalized with
other endothelium-specific proteins, vWf, and eNOS (images in the Merge
column also include DAPI nuclear stain). The presence and localization of CD38
was unchanged after ischemia or I/R. (C) A basal level of CD38 activity is
measured in control hearts; however, activity with ischemia and I/R is signifi-
cantly increased. **P < 0.01 versus Control (mean ± SEM; n = 5).
Fig. 5. Endothelial NADP(H) levels, correlation with NOS-dependent CF re-
covery, BH4 salvage, and NO production. Levels of endothelial NADP(H) were
determined by endothelial membrane permeabilization with measurement of
the effluent via HPLC. (A) ∼90% depletion of endothelial NADP(H) occurred
with I/R compared with control nonischemic hearts. CD38 inhibition with
α-NAD salvaged NADP(H). **P < 0.01 versus untreated I/R group (mean ± SEM;
n = 5–7). (B) Prevention of NADP(H) loss through CD38 inhibition by α-NAD
correlated with preservation of NOS-dependent CF. **P < 0.01 versus un-
treated I/R group (mean ± SEM; n = 5–7). (C) Levels of BH4 from whole-heart
tissue were depleted after 30 min of ischemia followed by 30 min of reper-
fusion. When 5 mM α-NAD was administered just before ischemia, the loss of
BH4 was blunted. **P < 0.01 versus untreated I/R group (mean ± SEM; n = 4).
(D) NO production from hearts was measured by EPR spin trapping. NO levels
dramatically decreased with I/R but were preserved with 5 mM α-NAD treat-
ment. **P < 0.01 versus untreated I/R group (mean ± SEM; n = 4).
Reyes et al. PNAS Early Edition | 3 of 6
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4. NADPH Supplementation and CD38 Inhibition Enhance Recovery of
Contractile Function and Decrease Infarction. To determine if the
normalization of eNOS-mediated endothelial function seen with
NADPH supplementation or CD38 inhibition is associated with
myocardial protection as evidenced by improved recovery of con-
tractile function and decreased infarction, additional studies were
performed in isolated hearts from WT and eNOS−/−
mice subjected
to I/R. In WT hearts, the recovery of both left ventricular (LV)
developed pressure (LVDP) and its peak derivative (dP/dt) were
∼75% higher with liposomal NADPH treatment (N) and ∼125%
higher with α-NAD treatment than in untreated hearts (Fig. 7).
Significantly lower LV end diastolic pressure (LVEDP) values
were seen, indicating improved diastolic relaxation. Infarct size was
also reduced by ∼30% in the treated WT hearts. In contrast, in
eNOS−/−
hearts there were no significant functional changes
with either treatment. In eNOS−/−
, NADPH treatment did not alter
infarction and α-NAD treatment exerted only a small decrease in
infarct size that did not reach significance, whereas in WT a signif-
icant decrease was seen (Fig. 7). Thus, the observed normalization
of endothelial function through NADPH supplementation or
CD38 inhibition is accompanied by myocardial protection.
Discussion
Loss of eNOS cofactors and substrates can trigger endothelial
dysfunction (13, 19). Our laboratory (10) and others (20) have
demonstrated that the NOS cofactor, BH4, is lost during ischemic
injury, contributing to endothelial dysfunction. Although BH4 re-
pletion can ameliorate endothelial dysfunction, the recovery is
incomplete (10). A recent clinical study highlighted the limitations
in recovery of endothelial function with BH4 repletion where
patients with coronary artery disease were unresponsive to BH4
treatment (11). The unsuccessful recovery of endothelial function
was reported to be due to the oxidative environment that ac-
companies coronary artery disease with oxidation of BH4 to BH2.
This inability to maintain BH4 in a reduced state suggests possible
impairment of NADPH-dependent BH4 salvage.
We observed that NADPH, the essential reducing substrate
for eNOS, was severely depleted during I/R and that this limited
eNOS activity in the heart. NADPH is required for reduction of
the FAD cofactor of eNOS, enabling catalytic conversion of
L-arginine and O2 to L-citrulline and NO (21). In addition,
NADPH is required for the function of the critical BH4 salvaging
enzyme, dihydrofolate reductase (22).
Measurements from heart homogenates revealed that NADPH is
depleted during I/R (Fig. 1). This loss of NADPH could not be
explained by oxidation, as levels of NADP+
decreased more than
NADPH. In contrast, the NAD(H) pool was much less affected.
This preferential depletion of NADP(H) as well as the more
marked NADP+
depletion relative to NADPH demonstrates that
the loss of NADP(H) cannot be explained by washout from dam-
aged cells; rather, this occurs due to a specific process that depletes
NADP+
with corresponding diminution of the NADP(H) pool.
The significance of this NADPH depletion on endothelial
function was demonstrated in studies in which repletion with li-
posomal NADPH resulted in marked restoration of postischemic
CF (Fig. 2A). Repletion of NADPH was ∼threefold more potent
than similar BH4 treatment, and combined NADPH and BH4
treatment achieved full restoration of NOS-dependent CF (Fig.
2B). The marked potency of NADPH in restoration of endothelial
vasodilatory function suggested that endothelial NADPH depletion
may be more severe than that in the myocardium. Measurements
of the endothelial NADP(H) pool demonstrated near total
depletion with I/R (Fig. 2C). Thus, I/R triggered severe deple-
tion of endothelial NADP(H), leading to severe impairment of
endothelium-dependent vasodilation.
Enzymes that catabolize pyridine nucleotides include poly-ADP
ribose polymerase (PARP), sirtuin deacetylases, and CD38 (22). Of
these, CD38 is unique in its substrate preference for NADP+
over
NAD+
with >sixfold lower Km and twofold higher Vmax (14). CD38
is a complex and multifunctional enzyme (Fig. 3) with wide distri-
bution in many tissues (14, 23–25). Originally identified as an antigen
marker on B cells (26), CD38 was later found to contain ADP
ribosyl cyclase, cADPR hydrolase, and NAD(P)ase activities. The
conversion of NADP+
to 2’-P-ADPR and NA can be achieved
through the cyclization of NADP+
to 2’-P-cADPR, followed by the
hydrolysis of 2’-P-cADPR to 2’-P-ADPR. CD38 can also act as an
NAD(P)ase, directly hydrolyzing NADP+
to 2’-P-ADPR and NA.
We observed that CD38 is present in the heart, with high levels of
expression in the endothelium (Fig. 4B). Although CD38 expression
did not change following the onset of I/R, its activity greatly in-
creased, accounting for the decrease in NADP+
levels with a cor-
responding increase in 2’-P-ADPR (Fig. 4C). The increased activity
of CD38 with ischemia and I/R was shown to cause the depletion of
the endothelial NADP(H), as inhibition of CD38 led to complete
salvage of endothelial NADP(H) and a near-complete recovery of
NOS-dependent CF and NO generation following I/R (Fig. 5 A, B,
and D). This was confirmed in studies of cultured endothelial cells
subjected to H/R where genetic knockdown of CD38 preserved the
levels of NADP(H) (SI Appendix, Fig. S9). Thus, CD38 activa-
tion was shown to have a profound effect on endothelial levels of
NADP(H) and eNOS function after I/R.
CD38 activation in the postischemic heart was inhibited by a SOD
mimetic, suggesting that O2
•–
can trigger activation. Thus, oxidative
modification of CD38 may contribute to its activation. CD38 has
essential cysteines that must be oxidized to form disulfide bonds for
maximum activity. Mutations of these cysteines decrease or abrogate
Fig. 6. Regulation of CD38 activity measured by 2’-P-ADPR levels. With activation
of CD38 activity by I/R, there is an increase in 2’-P-ADPR. The rise in 2’-P-ADPR level
was largely abolished by pretreatment with a SOD mimetic (MnTBAP), suggesting
activation was triggered by I/R-associated oxidant stress. The CD38 inhibitor α-NAD
decreased 2’-P-ADPR formation. **P < 0.01 versus control (mean ± SEM; n = 4).
Fig. 7. Effect of NADPH supplementation or CD38 inhibition on recovery of
contractile function and infarction in WT and eNOS−/−
hearts. Studies were
performed on isolated hearts that were pretreated with liposomal NADPH (N)
or the CD38 inhibitor (αNAD) and then subjected to I/R. Results are shown for
LVDP, maximum derivative of LVDP versus time (dP/dt), LVEDP, and infarct size
as percentage of the LV. In WT hearts, either NADPH or α-NAD treatment en-
hanced the recovery of contractile function, with higher LVDP and dP/dt seen
with lower LVEDP; whereas in eNOS−/−
no significant changes were seen with
either treatment. Liposomal NADPH decreased infarct size only in WT but not
in eNOS−/−
hearts. α-NAD decreased infarct size in WT with only a small ef-
fect in eNOS−/−
hearts that did not reach significance. **P < 0.01 and *P < 0.05
versus respective untreated group (mean ± SEM; n = 7–9).
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5. the enzymatic function of CD38 (27). It has also been reported that
endogenous or exogenous sources of O2
•–
can activate CD38 by
internalization of membrane-associated enzyme, leading to exposure
to its intracellular substrates, NADP+
and NAD+
(16, 18).
The activity of CD38 is typically measured by production of the
signaling molecules 2’-P-cADPR/cADPR, 2’-P-ADPR/ADPR, or
nicotinic acid adenine dinucleotide phosphate (NAADP) (14).
Both 2’-P-cADPR and cADPR signal Ca2+
release via intracellu-
lar stores (14). NAADP modulates lysosomal Ca2+
release, and
ADPR increases Ca2+
levels through activation of the transient
receptor potential cation channel, subtype M (28, 29). Because
Ca2+
overload is a major mechanism of postischemic injury, in-
hibiting the formation of these signaling molecules could confer
added benefit through moderation of Ca2+
release into the cy-
tosol in endothelial cells and cardiac myocytes.
Oxidative stress and Ca2+
overload are central mechanisms of
cardiac reperfusion injury. These two mechanisms interact and
potentiate each other with oxygen radicals inducing Ca2+
over-
load, which in turn activates pathways of oxygen radical gener-
ation (30–32). In prior reports, CD38 activity was shown to be
redox-sensitive, and exposure to oxidative stress and reactive
oxygen species is reported to increase CD38 activity (18, 24).
Consistent with this, we observed that treatment with a SOD
mimetic decreased postischemic CD38 activation with a 2.5-fold
decrease in levels of 2’-P-ADPR (Fig. 6).
Beyond its critical role in NOS function, NADPH is a requisite
substrate for a number of enzymes of central importance in re-
dox regulation and metabolism. NADPH provides the reducing
equivalents to protect against oxidative stress and is the substrate
regulating the glutathione redox state through glutathione reduc-
tase (33). It functions as a reducing substrate for dihydrofolate
reductase that is required for the de novo synthesis of purines,
thymidylic acid, and certain amino acids, in addition to serving
as the major BH4 salvage pathway (22). We observed that in-
hibition of CD38 with preservation of NADPH resulted in in-
creased levels of BH4, highlighting the importance of NADPH
for BH4 salvage in I/R injury (Fig. 5C).
It was observed that supplementation of NADPH or inhibition
of CD38 not only restored postischemic endothelial function but
also conferred myocardial protection with enhanced recovery of
contractile function and decreased infarction. This protection
was shown to be eNOS-dependent, as it was lost in eNOS−/−
hearts, suggesting that normalization of endothelial function can
result in generalized myocardial protection (Fig. 7).
In summary, we observe that the critical eNOS reducing substrate
NADPH is depleted in the postischemic heart. This loss is most
severe in the endothelium and occurs due to activation of the
NAD(P)ase function of CD38, thereby depleting the NADP(H) pool
and salvage of BH4, triggering eNOS dysfunction and uncoupling.
Thus, NADPH depletion is identified as a major cause of post-
ischemic endothelial dysfunction, with NADPH repletion or CD38
inhibition restoring normal endothelial vasodilation. This restoration
of endothelial function is particularly important, as it was also shown
to be accompanied by enhanced recovery of contractile function and
decreased infarction. These observations suggest that therapeutics
that restore NADPH and/or prevent its degradation may be benefi-
cial in the treatment of unstable coronary syndromes and myocardial
infarction. In view of the central importance of oxidant-induced
eNOS-mediated endothelial dysfunction in the pathogenesis of a
broad spectrum of disease, identification of this novel pathway pro-
vides critical therapeutic insights for disease prevention and treatment.
Methods
Isolated Heart Studies. Hearts from male, Sprague–Dawley rats (275–300 g) or
C57BL/6J mice (WT and eNOS−/−
) were prepared as described (10). eNOS−/−
mice,
strain B6.129P2-Nos3tm1Unc
/J, were purchased from Jackson Laboratory. Hearts
were excised, aorta cannulated, and perfused retrograde with Krebs buffer
[119 mM NaCl, 17 mM Glucose, 25 mM NaHCO3, 5.9 mM KCl, 1.2 mM MgCl2,
2.5 mM CaCl2, 0.5 mM EDTA, and 2 mM pyruvate (mice only)]. A balloon con-
nected to a pressure transducer was placed in the LV to measure contractile
function. An in-line flow probe and flowmeter (Transonic) were used to measure
CF. Measurements of infarct size were performed after 120 min of reperfusion
as described (34) (SI Appendix, SI Methods). Animal protocols were approved by
the Institutional Animal Care and Use Committee of The Ohio State University.
Materials. Unless otherwise noted, all chemicals as well as ADP ribosyl cyclase
from Aplysia californica were purchased from Sigma.
Experimental Protocols. For pyridine nucleotide measurements, hearts were
subjected to either control perfusion, periods of ischemia (10, 20, or 30 min),
or ischemia (30 min) followed by reperfusion (10, 20, or 30 min). Control
hearts were perfused for 60 min, whereas those undergoing global ischemia
or I/R were first perfused for a 20-min equilibration period. At the designated
time, hearts were freeze-clamped and stored in liquid nitrogen.
For measurements of CF restitution, hearts were subjected to 30 min of is-
chemia followed by 30 min of reperfusion and infused with either control li-
posomes (25 mg/mL), liposomal NADPH (175 μM), liposomal BH4 (50 μM),
liposomal NADPH (175 μM) + BH4 (50 μM), or liposomal NADPH (175 μM) +
L-NAME (1 mM) for 10 min in Krebs buffer.
An additional set of hearts was used to measure endothelial NADP(H) after
30 min of ischemia followed by 30 min of reperfusion. Endothelial per-
meabilization was performed using the procedure of Giraldez et al., which
selectively permeabilizes the endothelium, abrogating endothelial function,
with minimal effects on cardiac myocytes (13). At the desired time, hearts
received a brief (∼1 s) bolus of 0.25% Triton X-100, permeabilizing the en-
dothelium allowing washout of endothelial substrates including the NADP(H)
pool. The effluent was collected for three 1-min intervals. Additional hearts re-
ceived either α-NAD (5 mM) or vehicle (Krebs buffer) infused for 3 min before
the onset of ischemia. After 30 min of reperfusion, endothelial permeabilization
was performed and effluent collected.
A final set of hearts was used to measure the effect of oxidant stress on
2’-P-ADPR production. Before ischemia, hearts were administered either
α-NAD or the SOD mimetic MnTBAP (30 μM) for 3 min. Following 30 min of
ischemia and 30 min of reperfusion, hearts were prepared for HPLC.
Detection of Pyridine Nucleotides by HPLC. NAD(H) and NADP(H) detection
was performed in hearts by modification of the method of Klaidman et al.
(35) (SI Appendix, SI Methods). Samples were injected onto a TSKgel ODS-
80TM column (25 cm × 4.6 mm) (Supelco). Separation was achieved with a
flow rate of 1.0 mL/min and methanol gradient (0.2%/min for 25 min).
Analytes were detected via fluorescence (ex. 330 nm; em. 460 nm).
Detection of Pyridine Nucleotides Following Endothelial Permeablization. Be-
fore HPLC analysis, the heart effluent absorption spectrum was measured (Cary 50
Bio UV/VIS Spectrophotometer, Varian) and demonstrated strong absorption at
260 nm and 340 nm [for NAD(P)+
and NAD(P)H, respectively], with a lack of
absorption from the α/β bands of heme, confirming a lack of myocyte leakage of
myoglobin. Triton X-100 was removed from samples with addition of Surfact-
away (BioTech Support Group) in a 1:4 dilution. Samples were concentrated using
a SpeedVac (Thermo Scientific) and resuspended in 250 μL of potassium cyanide
(KCN) buffer (KCN 0.2 M, KOH 0.06 M, and DTPA 1 mM). Surfactaway was again
added at 1:4 dilution to ensure complete removal of Triton X-100, concentrated,
and then HPLC measurements were performed as described above.
Immunoblotting for CD38. Hearts were homogenized in 150 mM NaCl, 10 mM
Tris, 1 mM EDTA with protease inhibitors, and 1% Triton X-100. Homogenates
were separated on 10% (wt/vol) Tris-glycine polyacrylamide gels. Protein from
gels was transferred to nitrocellulose membranes and blocked for 1 h at room
temperature (RT) in Tris-buffered saline containing 0.075% Tween-20 (TBST),
with 5% (wt/vol) milk. Membranes were incubated overnight with anti-
CD38 antibody (Santa Cruz, M-19) diluted at 1:1,000 at 4 °C. Membranes were
washed in TBST and incubated for 1 h with HRP-conjugated anti-rabbit IgG
in TBST at RT. Imaging was performed with ECL immunoblotting detection
reagents (Amersham Biosciences) and bands quantified with NIH ImageJ.
Membranes were stripped and similarly immunoblotted with an anti-GAPDH
antibody (Cell Signaling) diluted to 1:10,000.
Immunohistochemistry of CD38, vWF, and eNOS. Isolated hearts were embed-
ded in optimal cutting temperature compound and sectioned using a cryotome
at –20 °C. Sections (5 μm) were attached to coverslips, fixed with 3.7% (wt/vol)
paraformaldehyde (10 min), permeabilized with 0.25% Triton X-100 in TBST
(0.01% Tween; 5 min), and blocked for 30 min with 1% BSA in TBST and in-
cubated with anti-CD38 (Santa Cruz Biotechnology), anti-eNOS (BD Biosciences),
and anti-vWF (Abcam) primary antibodies at dilutions of 1:300, 1:500, and 1:200,
respectively, in TBST containing 1% BSA (1 h; RT). Sections were then incubated
Reyes et al. PNAS Early Edition | 5 of 6
MEDICALSCIENCES

6. with secondary antibodies: Alexa Fluor 488 (anti-goat), 568 (anti-mouse), and 647
(anti-rabbit) (Life Technologies) at 1:1,000 dilution (1 h; RT). Nuclei were stained
with 4’,6-diamidino-2-phenylindole (DAPI). After washing with TBST, the sections
were mounted with Fluoromount-G (Southern Biotech). Slides were visualized
using an Olympus FV1000 imaging system.
HPLC Measurement of BH4. Hearts were homogenized in ice-cold 0.1 N HCl. BH4
and BH2 were then oxidized to biopterin and pterin using a 2% (wt/vol) KI/
3% (wt/vol) iodine solution in acid or basic conditions. Samples were loaded
onto an Atlantis T3 reverse phase column (5 μm; 4.6 × 150 mm) (Waters
Corporation). Isocratic elution was performed at a flow rate of 1.2 mL/min,
using a buffer consisting of 100 mM KH2PO4 (pH 2.5), 6 mM citric acid, 2.5 mM
1-octanesulfonic acid (OSA), and 2% (vol/vol) methanol (9). Analytes were de-
tected via fluorescence spectroscopy (wavelength ex. 348 nm; em. 444 nm).
HPLC Measurement of 2’-P-ADPR Levels and CD38 Activity. Hearts were ho-
mogenized in ice-cold buffer (Hepes 20 mM, pH 7.3, Sucrose 250 mM, EDTA
0.1 mM, β-2 Mercaptoethanol 2.5 mM), and samples were subjected to
chloroform treatment (1:1 volume) followed by centrifugation at 16,000 × g
(2×; 5 min). The aqueous phase was collected and filtered using a Costar
Spin-X 0.45 μm pore size filter. The effluent from Costar Spin-X tubes was
diluted 1:3 with mobile phase (0.04 M Sodium Phosphate/1% methanol, pH
7.0). Filtrate was injected onto a TSKgel ODS-80TM column (25 cm × 4.6 mm)
(Supelco) and separation achieved as described with UV/Vis detection at 254
nm (36) (SI Appendix, SI Methods). 2’-P-ADPR standard was prepared by in-
cubating NADP+
with ADP ribosyl cyclase of A. californica. CD38 activity was
measured by in vitro assays of 2’-P-ADPR formation. After homogenization,
samples were centrifuged at 16,000 × g (2×; 5 min) at 4 °C. The super-
natants were kept on ice and a HiTrap desalting column used to remove
enzyme substrate. We then added 25 μL of homogenate (∼60 μg protein) to
the reaction mixture (Tris buffer 50 mM, pH 7.4 with 100 μM NADP+
), with a
10-min incubation at 37 °C. Upon reaction completion, samples were placed on
ice and centrifuged using Costar Spin-X 0.45 μm pore size filters (3 min). HPLC
measurements of 2’-P-ADPR were performed on the filtrate.
Measurements of NOS Activity. NOS activity was measured by L-[14
C]arginine
to L-[14
C]citrulline conversion assay as previously reported (10, 19) (SI Ap-
pendix, SI Methods).
EPR Spin Trapping of NO and O2
•–
. EPR spin trapping of NO was performed
using Fe-MGD (0.5 mM, 2.5 mM) in perfusate with 10 mg/mL BSA. Spin
trapping of O2
•–
was performed using 50 mM DMPO in perfusate with heart
effluent immediately mixed with 100 mM methyl-β-cyclodextrin. Measure-
ments were performed with and without SOD1 (200 U/mL) or L-NAME (1 mM)
(13) using a Bruker EMX spectrometer (SI Appendix, SI Methods).
Stable Knockdown of CD38 in RAECs. Stable knockdown of CD38 was per-
formed using shRNA plasmid provided by Hon-Cheung Lee, University of
Hong Kong, Hong Kong (37), as described in SI Appendix, SI Methods.
H/R Model. RAECs were washed with PBS and kept in serum-free DMEM in a
hypoxic environment created by placing the flasks containing cells at con-
fluence into a Billups–Rothenberg incubator chamber flushed with a 95%
N2/5% CO2 gas as detailed in SI Appendix, SI Methods.
Statistics. Results were expressed as mean ± SE. Statistical significance was
determined by ANOVA (followed by Newman–Keuls test) for multiple groups.
Paired or unpaired t tests were used for comparison between two groups.
ACKNOWLEDGMENTS. This work was supported by NIH Grants HL63744,
HL65608, HL38324, and EB016096 (to J.L.Z.).
1. Yetik-Anacak G, Catravas JD (2006) Nitric oxide and the endothelium: History and
impact on cardiovascular disease. Vascul Pharmacol 45(5):268–276.
2. Mehta JL, Nichols WW, Donnelly WH, Lawson DL, Saldeen TG (1989) Impaired canine coronary
vasodilator response to acetylcholine and bradykinin after occlusion-reperfusion. Circ Res
64(1):43–54.
3. VanBenthuysen KM, McMurtry IF, Horwitz LD (1987) Reperfusion after acute coronary oc-
clusion in dogs impairs endothelium-dependent relaxation to acetylcholine and augments
contractile reactivity in vitro. J Clin Invest 79(1):265–274.
4. Bolli R (1990) Mechanism of myocardial “stunning”. Circulation 82(3):723–738.
5. Zweier JL, Flaherty JT, Weisfeldt ML (1987) Direct measurement of free radical generation
following reperfusion of ischemic myocardium. Proc Natl Acad Sci USA 84(5):1404–1407.
6. Palmer RM, Ferrige AG, Moncada S (1987) Nitric oxide release accounts for the bi-
ological activity of endothelium-derived relaxing factor. Nature 327(6122):524–526.
7. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G (1987) Endothelium-derived re-
laxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci
USA 84(24):9265–9269.
8. Xia Y, Dawson VL, Dawson TM, Snyder SH, Zweier JL (1996) Nitric oxide synthase
generates superoxide and nitric oxide in arginine-depleted cells leading to peroxy-
nitrite-mediated cellular injury. Proc Natl Acad Sci USA 93(13):6770–6774.
9. De Pascali F, Hemann C, Samons K, Chen CA, Zweier JL (2014) Hypoxia and reoxygenation
induce endothelial nitric oxide synthase uncoupling in endothelial cells through tetra-
hydrobiopterin depletion and S-glutathionylation. Biochemistry 53(22):3679–3688.
10. Dumitrescu C, et al. (2007) Myocardial ischemia results in tetrahydrobiopterin (BH4)
oxidation with impaired endothelial function ameliorated by BH4. Proc Natl Acad Sci
USA 104(38):15081–15086.
11. Cunnington C, et al. (2012) Systemic and vascular oxidation limits the efficacy of
oral tetrahydrobiopterin treatment in patients with coronary artery disease.
Circulation 125(11):1356–1366.
12. Velayutham M, Li H, Kuppusamy P, Zweier JL (2003) Mapping ischemic risk region and
necrosis in the isolated heart using EPR imaging. Magn Reson Med 49(6):1181–1187.
13. Giraldez RR, Panda A, Zweier JL (2000) Endothelial dysfunction does not require loss of
endothelial nitric oxide synthase. Am J Physiol Heart Circ Physiol 278(6):H2020–H2027.
14. Vu CQ, Lu PJ, Chen CS, Jacobson MK (1996) 2′-Phospho-cyclic ADP-ribose, a calcium-
mobilizing agent derived from NADP. J Biol Chem 271(9):4747–4754.
15. Okayama H, Ueda K, Hayaishi O (1980) NAD glycohydrolases from rat liver nuclei.
Methods Enzymol 66:151–154.
16. Xu M, et al. (2013) Contribution of NADPH oxidase to membrane CD38 internalization
and activation in coronary arterial myocytes. PLoS One 8(8):e71212.
17. Zhang G, et al. (2006) Cyclic ADP ribose-mediated Ca2+ signaling in mediating en-
dothelial nitric oxide production in bovine coronary arteries. Am J Physiol Heart Circ
Physiol 290(3):H1172–H1181.
18. Zhang AY, Yi F, Teggatz EG, Zou AP, Li PL (2004) Enhanced production and action of cyclic
ADP-ribose during oxidative stress in small bovine coronary arterial smooth muscle. Microvasc
Res 67(2):159–167.
19. Giraldez RR, Panda A, Xia Y, Sanders SP, Zweier JL (1997) Decreased nitric-oxide synthase
activity causes impaired endothelium-dependent relaxation in the postischemic heart.
J Biol Chem 272(34):21420–21426.
20. Masano T, et al. (2008) Beneficial effects of exogenous tetrahydrobiopterin on left ven-
tricular remodeling after myocardial infarction in rats: The possible role of oxidative stress
caused by uncoupled endothelial nitric oxide synthase. Circ J 72(9):1512–1519.
21. Panda K, Adak S, Konas D, Sharma M, Stuehr DJ (2004) A conserved aspartate (Asp-
1393) regulates NADPH reduction of neuronal nitric-oxide synthase: Implications for
catalysis. J Biol Chem 279(18):18323–18333.
22. Armarego WL, Randles D, Taguchi H (1983) Peroxidase catalysed aerobic degradation
of 5,6,7,8-tetrahydrobiopterin at physiological pH. Eur J Biochem 135(3):393–403.
23. Thai TL, Arendshorst WJ (2009) Mice lacking the ADP ribosyl cyclase CD38 exhibit
attenuated renal vasoconstriction to angiotensin II, endothelin-1, and norepineph-
rine. Am J Physiol Renal Physiol 297(1):F169–F176.
24. Chidambaram N, Wong ET, Chang CF (1998) Differential oligomerization of membrane-
bound CD38/ADP-ribosyl cyclase in porcine heart microsomes. Biochem Mol Biol Int 44(6):
1225–1233.
25. Cockayne DA, et al. (1998) Mice deficient for the ecto-nicotinamide adenine dinucleotide
glycohydrolase CD38 exhibit altered humoral immune responses. Blood 92(4):1324–1333.
26. Reinherz EL, Kung PC, Goldstein G, Levey RH, Schlossman SF (1980) Discrete stages of
human intrathymic differentiation: Analysis of normal thymocytes and leukemic
lymphoblasts of T-cell lineage. Proc Natl Acad Sci USA 77(3):1588–1592.
27. Zhao YJ, Zhang HM, Lam CM, Hao Q, Lee HC (2011) Cytosolic CD38 protein forms intact
disulfides and is active in elevating intracellular cyclic ADP-ribose. J Biol Chem 286(25):
22170–22177.
28. Churchill GC, et al. (2002) NAADP mobilizes Ca(2+) from reserve granules, lysosome-
related organelles, in sea urchin eggs. Cell 111(5):703–708.
29. Yamasaki M, et al. (2004) Organelle selection determines agonist-specific Ca2+ signals
in pancreatic acinar and beta cells. J Biol Chem 279(8):7234–7240.
30. Josephson RA, Silverman HS, Lakatta EG, Stern MD, Zweier JL (1991) Study of the
mechanisms of hydrogen peroxide and hydroxyl free radical-induced cellular injury
and calcium overload in cardiac myocytes. J Biol Chem 266(4):2354–2361.
31. Corretti MC, et al. (1991) Glycolytic inhibition and calcium overload as consequences
of exogenously generated free radicals in rabbit hearts. J Clin Invest 88(3):1014–1025.
32. Talukder MA, Zweier JL, Periasamy M (2009) Targeting calcium transport in ischaemic
heart disease. Cardiovasc Res 84(3):345–352.
33. Burgoyne JR, Mongue-Din H, Eaton P, Shah AM (2012) Redox signaling in cardiac
physiology and pathology. Circ Res 111(8):1091–1106.
34. Talukder MAH, et al. (2008) Is reduced SERCA2a expression detrimental or beneficial
to postischemic cardiac function and injury? Evidence from heterozygous SERCA2a
knockout mice. (Translated from English). Am J Physiol-Heart C 294(3):H1426–H1434.
35. Klaidman LK, Leung AC, Adams JD, Jr (1995) High-performance liquid chromatogra-
phy analysis of oxidized and reduced pyridine dinucleotides in specific brain regions.
Anal Biochem 228(2):312–317.
36. Broetto-Biazon AC, et al. (2008) Transformation and actions of extracellular NADP(+)
in the rat liver. Mol Cell Biochem 317(1-2):85–95.
37. Yue J, et al. (2009) CD38/cADPR/Ca2+ pathway promotes cell proliferation and delays
nerve growth factor-induced differentiation in PC12 cells. J Biol Chem 284(43):
29335–29342.
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